The long-term goal of the proposed studies is to define the molecular basis of insulin action on unconventional myosin motors and the actin cytoskeleton within the context of how these elements facilitate glucose transporter regulation by insulin. GLUT4 glucose transporters mediate the important physiological effect of insulin to stimulate glucose transport into fat and muscle in a PI 3-kinase dependent manner. Recent work shows a requirement of actin filaments for optimal translocation of GLUT4-containing vesicles to the plasma membrane of both primary and cultured adipocytes in response to insulin. Insulin itself causes acute polymerization of F-actin in these cells, apparently through a PI 3-kinase independent pathway. These and other data have spawned the hypothesis that at least two pathways are required in tandem for optimal insulin action on GLUT4 movements: A PI 3-kinase-dependent signaling cascade through Akt and perhaps other downstream effectors, and a PI 3-kinase-independent mechanism that modulates actin dynamics required to facilitate GLUT4-containing vesicle movements to the plasma membrane. Novel results from our laboratory reveal a potential role of the actin cytoskeleton in directly guiding GLUT4 movements near the plasma membrane via the actions of unconventional myosins such as Myo1c. We found that Myo1c is highly expressed in adipocytes, is recruited to peripheral actin filaments and GLUT4 vesicles by insulin in cultured adipocytes by a PI 3-kinase-independent mechanism, and appears to be required for GLUT4 recruitment to the plasma membrane. This proposal exploits these new findings using unique techniques we have recently developed for assessing the function of proteins in GLUT4 recycling. We shall test the hypothesis that Myo1c function is required for GLUT4 movements by selectively ablating its expression in cultured adipocytes with interference RNA techniques (siRNA) and morpholino antisense RNA constructs, and in primary mouse fat and muscle by conditional gene knockout methodology. We shall also test the hypothesis that Myo1c directly connects GLUT4 vesicles to F-actin and moves GLUT4-containing vesicles along F-actin filaments by: 1. assessing the distance between cortical Myo1c molecules and GLUT4 in intact cells using FRET and 2. developing a cell free system for studying GLUT4-containing vesicle movements on actin filaments, analogous to that developed for synaptic vesicles. We shall also test the hypothesis that insulin signaling causes recruitment of Myo1c to actin filaments secondary to its ability to cause F-actin polymerization. The idea that this occurs through the activation of N-WASP or similar proteins via the adaptor Nck will be further tested by ablating expression of these proteins in adipocytes. Together, these experiments will clarify the role of Myo1c and the actin cytoskeleton in GLUT4 regulation by insulin.